The general objective of the research described in this application is to elucidate the mechanism of regulation of microtubule assembly in vitro and in vivo at the level of the tubulin molecule. The protein tubulin is an alpha-beta heterodimer which is the structural subunit of the microtubule. The fact that microtubules are involved in many processes including mitosis and secretion means that regulation of microtubule assembly is important in understanding these processes. In order to control uncontrolled cell division, as in cancer, it is necessary to understand the regulation of normal cell division. Our approach will be to focus on the sulfhydryls of tubulin, the in vivo phosphorylation of tubulin, and the role of tubulin isotypes. In Specific Aim 1 we will identify the sulfhydryls that are located close to or at the binding sites on tubulin of the anti-tumor drugs vinblastine and maytansine as well as of the normal in vivo ligand GTP. In Specific Aim 2 we will test the hypothesis that the beta 2 isotype of tubulin is a substrate for in vivo phosphorylation and will attempt to identify the phosphorylated residues. We will also investigate the effect of phosphorylation on the in vitro properties of tubulin. In Specific Aims 3 and 4 we will prepare monoclonal antibodies able to discriminate between the beta 1 and beta 2 isotypes and use them to purify alpha-beta 1 and alpha-beta 2 tubulin dimers in their native states to study their structural and functional properties in vitro. In Specific Aim 5 we will use these antibodies in immunohistochemical assays to localize the beta 1 and beta 2 isotypes in vivo. In Specific Aim 6 the possible roles of the sulfhydryl proteins thioredoxin and thioredoxin reductase in regulating microtubule assembly will be examined.